Enzymatic C−H Oxidation–Amidation Cascade in the Production of Natural and Unnatural Thiotetronate Antibiotics with Potentiated Bioactivity

The selective activation of unreactive hydrocarbons by biosynthetic enzymes has inspired new synthetic methods in C−H bond activation. Herein, we report the unprecedented two‐step biosynthetic conversion of thiotetromycin to thiotetroamide C involving the tandem oxidation and amidation of an unreactive ethyl group. We detail the genetic and biochemical basis for the terminal amidation in thiotetroamide C biosynthesis, which involves a uniquely adapted cytochrome P450–amidotransferase enzyme pair and highlights the first oxidation–amidation enzymatic cascade reaction leading to the selective formation of a primary amide group from a chemically inert alkyl group. Motivated by the ten‐fold increase in antibiotic potency of thiotetroamide C ascribed to the acetamide group and the unusual enzymology involved, we enzymatically interrogated diverse thiolactomycin analogues and prepared an unnatural thiotetroamide C analogue with potentiated bioactivity compared to the parent molecule.